J.L. La Torre

Large-scale use of liquid-phase blocking sandwich ELISA for the evaluation of protective immunity against aphthovirus in cattle vaccinated with oil-adjuvanted vaccines in Argentina

Specific serum activity levels against four reference strains of foot-and-mouth disease virus (FMDV) were evaluated from 1634 animals vaccinated with commercial quadrivalent oil vaccines and from 746 unvaccinated, naive animals, using the liquid-phase blocking sandwich ELISA (lpELISA) test. Cows from the FMDV-free area of Argentina were tested for the absence of specific FMDV antibodies (sp FMDV Abs) and those showing lpELISA titres < 1.0 were grouped in lots of 16 animals. They were vaccinated and challenged at 90 days postvaccination (DPV) with one of four virus strains used for vaccine production and control (prototype strains). Serum samples from vaccinated and control cattle were collected 60 and 90 DPV and the level of sp FMDV Abs was determined by lpELISA. Animals were examined for clinical signs of disease. Results show that serum lpELISA titre levels directly correlate with the percentage of protected animals. It was seen that 100, 98, 93 and 87% of the vaccinated cattle with antibody titre levels > or = 2.1 were protected against challenge with serotypes C85, A87,01 Cas and A79, respectively. Evidence is also presented of seroconversion in a sample of 3-5-month-old calves vaccinated in the field, showing lpELISA titres compatible with protection against the four vaccine viruses as long as 150 DPV. Results reported in this paper strongly support the use of the lpELISA test for a rapid and reliable evaluation of the efficacy of FMDV commercial vaccines as well as for the assessment of the immunological status of cattle in FMDV-free and enzootic regions of South America.

Characterization of Triatoma Virus, a Picorna-like Virus Isolated from the Triatomine Bug Triatoma infestans

Some properties of Triatoma virus (TrV), a picorna-like virus recently isolated from Triatoma infestans, have been studied. Electron microscopic observations of purified viral preparations showed the presence of non-enveloped viral particles 30 nm in diameter. The sedimentation coefficient of virus particles was about 165S and the buoyant density in CsCl was 1.39 g/ml. The viral genome was composed of one single-stranded RNA molecule with an Mr of 3 x 10(6). Three major polypeptides with Mr values of 39K, 37K and 33K and a minor one of about 45K were found in the virus particle. TrV particles contain about 35% RNA and 65% protein by weight. These data support the classification of this virus in the family Picornaviridae.

Assessment of foot and mouth disease vaccine potency by liquid-phase blocking ELISA: a proposal for an alternative to the challenge procedure in Argentina

The lowest expected protection (LEP) at a 95% confidence of 245 foot and mouth disease (FMD) commercial vaccines was calculated from the titres of liquid-phase blocking sandwich ELISA (lpELISA) of cattle sera obtained from 3920 animals at 60 days post-vaccination (d.p.v.) and challenged with live virus at 90 d.p.v. It was found that LEP evaluation is highly specific (i.e. it is able to predict the failure in 100% of the cases) although its ability to predict the challenge (PG test) approval (i.e. sensitivity) comprised only 65% of the vaccines that passed the trial. It was possible, nevertheless, to improve the sensitivity of the evaluation by using an alternative coefficient (Ro), exclusively dependent on the number of animals exhibiting the highest and lowest lpELISA titres in a particular vaccine trial. This coefficient was capable of predicting the PG approval of 90% of the vaccines, yet maintaining acceptable levels of safety (87% of specificity). Based on these results and as a first step towards the replacement of the challenge protocol in Argentina, we propose a swift approval for commercialization of FMD vaccines which are able to reach the highly restricting LEP passmark of 82%, and the rejection of those not reaching the 50% LEP limit. More extensive experience with this new protocol will allow a finer adjustment of the LEP and Ro values and to set more precisely the cut-off points for direct approval or disapproval of vaccines by lpELISA, eliminating the use of live FMDV in the field.

Total and isotype humoral responses in cattle vaccinated with foot and mouth disease virus (FMDV) immunogen produced either in bovine tongue tissue or in BHK-21 cell suspension cultures

The anti-foot and mouth disease virus (FMDV) serum antibody activity of protected and non protected animals immunized with inactivated FMDV originated in either bovine tongue tissue (BTTV vaccines) or BHK-21 cell suspension cultures (BHKV vaccines) was evaluated. The results show that 80-100% of the BTTV immunized and only 40-60% of the BHKV immunized animals with liquid-phase blocking sandwich ELISA (lp ELISA) serum titres of 1.5-1.7 U, were protected against the challenge with any of the four infectious FMDV argentine reference strains. This difference becomes almost marginal among BTTV and BHKV vaccinated animals with a strong anti-FMDV humoral response (i.e. lp ELISA titres > or = 1.95 U). Isotyping of the anti-FMDV response in immunized cattle with low lp ELISA titres revealed that BTTV vaccines were able to induce remarkably higher anti-FMDV IgG1 titres than their BHKV counterparts (i.e. mean titres of 1.95 and 1.35 U. respectively). This difference in specific IgG1 serum levels induced by BTTV and BHKV vaccines seems to be also limited to those animals with low anti-FMDV lp ELISA titres. These results together with the fact that the specific serum IgG1, but not the IgG2, isotype response of 219 vaccinated animals correlates almost linearly with their capacity to pass the challenge, suggests that the superior performance of BTTV vaccines is close related to their ability to raise a stronger anti-FMDV IgG1 response than BHKV vaccines.

Expression of foot and mouth disease virus non-structural polypeptide 3ABC induces histone H3 cleavage in BHK21 cells.

Auto-processing of the non-structural polypeptide 3ABC of foot and mouth disease virus (FMDV) expressed in Escherichia coli-BL21-DE3 was prevented by mutating either four glutamic acid residues at the 3A/3B1, 3B1/2, 3B2/3 and 3B3/3C junctions (3ABCtet) or a single cysteine residue at position 383 within the 3C domain (3ABCm). Independent expression of 3ABC and 3ABCtet genes induced expression of chaperone DnaK and degradation of ribosomal S1 protein in E. coli. They also induced cleavage of nucleosomal histone H3 when transiently expressed in BHK21 cells. 3ABCtet, 3ABCm, 3AB and 3A proteins concentrated in the perinuclear region suggesting that peptide sequences within the 3A domain specify intracellular targeting of 3ABC in BHK-21 cells. We propose that 3ABC molecules localized in the nuclear periphery are a source of protease 3C activity and are responsible for histone H3 processing during FMDV infections.

Rearrangement of Genomic Segment 11 in Two Swine Rotavirus Strains

We have recently reported the isolation of two group A swine rotaviruses each lacking normal genomic RNA segment 11 and showing instead one extra segment that migrated abnormally on gel electrophoresis. Hybridization studies performed with segment-specific probes and with a purified abnormal RNA segment showed that the extra bands had sequence homology to normal segment 11. Analysis of protein profiles of normal and rearranged strains showed that the gene product of segment 11 had no apparent change in its relative electrophoretic migration, suggesting that the rearranged genes remained functional.

Efficacy of an inactivated oil-adjuvanted rotavirus vaccine in the control of calf diarrhoea in beef herds in Argentina

Abstract We have assessed the potency of an inactivated oil-adjuvanted rotavirus vaccine in beef herds in Argentina. Two different vaccine trials were conducted. In a small-scale experimental trial, involving 21 pregnant cows (13 vaccinated and eight unvaccinated controls), a significant increase in neutralizing antibody titres against different serotypes of bovine rotaviruses was found in both the colostrum and serum of vaccinated cows compared with that of unvaccinated controls. Seven days after birth, half of the calves born to vaccinated dams or to control cows were challenged with live virulent virus whereas the other half of both groups were left in contact with the infected calves in order to mimic a natural field challenge. Although no statistically significant differences in the rate of protection were observed among the different groups of animals, a larger number of vaccinated calves were protected in comparison with their controls, particularly where animals in contact with infected calves were concerned. Secondly, a large-scale field trial was carried out in 17 beef herds involving a total of 4066 vaccinated pregnant cows. In 11 farms morbidity and mortality in calves from vaccinated cows were compared with historical data from the previous 3 years at the same locations. In the other six herds, control groups were used to compare data of the same year: 1540 cows were vaccinated and 2700 were left as controls. Taking into account the previous and current incidence of diarrhoea, morbidity and mortality were significantly reduced in 16 of the 17 beef herds tested. Vaccine effectiveness was also evident in farms where other enteropathogens such as cryptosporidium and coronaviruses were present, together with rotavirus.

Heterogeneity of the polyribocytidilic acid tract in aphthovirus: changes in the size of the poly(C) of viruses recovered from persistently infected cattle

A sample of aphthovirus type C3 strain Resende carrying two polyribocytidilic acid [poly(C)] tracts was cloned in tissue culture. One clone with a poly(C)-rich tract of about 145 nucleotides long (clone 3B) and another with a poly(C)-rich tract of about 230 nucleotides long (clone 12) and a mixture of both were injected intralingually into three steers. Samples from all three animals were recovered during the acute phase of the disease, from the blood and from the feet, and at various days after inoculation from the oesophageal-pharyngeal (OP) fluids. Analysis of the viral RNAs of the positive samples by means of RNase T1 maps on one- and two-dimensional gels showed (1) changes in the electrophoretic mobility of the poly(C)-rich tracts of viruses recovered from the OP fluids at various times after infection; (2) selection of virus populations with poly(C)-rich tracts of increased size; (3) later on, changes in the patterns of oligonucleotides of persistent viruses. These variations may lead to the production of new strains with altered biological properties that may contribute to the maintenance and spread of these viruses in the field.

Genome rearrangements in porcine rotaviruses: biochemical and biological comparisons between a supershort strain and its standard counterpart.

Two porcine rotavirus strains (CN86 and CC86) isolated during an epidemiological survey of diarrhoea in swine in Argentina were studied because of several unique characteristics. Both these strains were isolated and cloned from the same faecal sample and the electrophoretic migration of 10 of their 11 genomic dsRNA genomic segments in polyacrylamide gels was identical, but strain CC86 had a supershort electropherotype. We analysed biochemical, serological and biological properties of both viruses. In vitro translation of genome segment 11 RNAs showed that both viruses produced a polypeptide with an apparent Mr of 26K. No differences in any of the other virus-induced proteins made in infected MA104 cells were found on one- and two-dimensional gels for either strain. In addition, the serotype and the subgroup specificities of both viruses were identical (group A, subgroup I, serotype 5). These results suggest that the rearranged strain was probably generated from the standard one and that the coding capacity of the rearranged segment was conserved. Consistent with this hypothesis, primer extension analysis revealed that the supershort strain had a rearrangement involving partial duplication of genomic segment 11. Biological studies showed differences between these viruses. The rearranged strain (CC86) produced larger plaques in monolayers of MA104 cells and outgrew the standard strain (CN86) when cells were coinfected with both viruses at different relative concentrations and different m.o.i. The possibility that large plaque formation and efficient virus replication can be influenced by the products of genomic segment 11, in addition to segment 4, is discussed.

Inactivation of foot-and-mouth disease virus vaccine strains by activation of virus-associated endonuclease.

A new inactivation process for foot-and-mouth disease virus (FMDV) has been developed. This process is based on the activation of the FMDV endonuclease by incubation of unfractionated viral suspension or purified virions at 37 degrees C in the presence of high concentrations of monovalent cations such as K+, Cs+ or NH4+ at pH 8.5. This procedure completely inactivated several FMDV vaccine strains yielding preparations having similar amounts of 140S particles to untreated controls. The inactivation followed first-order kinetics and the rate of inactivation was faster than that achieved with other agents, e.g. binary ethyleneimine. Testing in suckling mice or tissue culture revealed no residual infectivity after inactivation. Virus particles purified from inactivated preparations showed (i) the same sedimentation coefficient as non-inactivated preparations, (ii) electrophoretic patterns of their viral capsid proteins identical to those derived from non-inactivated preparations, and (iii) extensive degradation of the 35S viral RNA. This method is safer than inactivation with aziridines because only innocuous chemicals are used in the process.